Diet, especially a high-fat diet, is of great concern to all Americans, for it has far-reaching effects on our health and well-being. Obesity and hyperlipidemia are major health factors affecting the body's susceptibility to a variety of diseases, such as heart attack, stroke, and diabetes. Long-chain fatty acids are the main constituents of fats and lipids. Humans, like other animals, derive their fatty acids from dietary sources and by de novo synthesis from acetyl-CoA, malonyl-CoA, and NADPH, a reaction catalyzed by the multifunctional enzyme, fatty acid synthase (FAS). The long range goal of the proposed research program is to provide detailed information, at the molecular level, of the mechanisms involved in the biosynthesis and regulation of fatty acids. The proposed studies include investigations of the structure-function of the multifunctional human FAS (hFAS). In humans, the liver is the main site of fatty acid synthesis. Starvation or diabetes decrease and refeeding or hormones, such as insulin and triiodothyronine (T3), increase fat production by increasing the activity of animal liver FAS, most likely by regulating FAS gene expression. Recently, FAS has been used successfully as a prognostic molecule in identifying the tumor cells of patients with breast cancer, who have a markedly worse prognosis. Breast tumors marked with high levels of FAS that do not respond to diet are four times more likely to recur and metastasize than those not so marked. Hence, studies of the structure-function of hFAS and the regulatory regions of its gene are of great value to an understanding of fat metabolism and regulation in humans. Until recently, knowledge of FAS has been garnered from studies of the enzyme in animal tissues, bacteria, and yeast. More can be learned about hFAS now because the enzyme has been isolated in a highly purified state from HepG2 cells, a well-differentiated human hepatoblastoma cell line. Also, the hFAS cDNA has been cloned and sequenced. The hFAS cDNA, which encodes 2504 amino acids, has been cloned and expressed in a bacterial host system as a soluble and active enzyme. The successful expression of hFAS cDNA in bacteria would afford an opportunity to not only express and prepare hFAS and its component enzymes in greater quantities, but also to carry out site-directed mutagenesis to probe the mechanism of action of the active centers of the FAS component enzymes and, ultimately, to reconstitute the FAS complex.
